Detecting RNA-protein proximity at DNA double-strand breaks using combined fluorescence in situ hybridization with proximity ligation assay

Summary RNA transcribed at DNA double-strand breaks (DSBs) contributes to accurate DNA repair. Here, using the repair factors 53BP1 and TIRR as examples, we combine the fluorescence in situ hybridization (FISH) and proximity ligation assay (PLA) techniques to determine protein proximity to DSB-transcribed RNA. In this FISH-PLA protocol, we detail steps for designing DNA probes and image analysis using CellProfiler™ software. This approach has many potential applications for the study of the RNA-binding proteins and nascent RNA interactions. For complete details on the use and execution of this protocol, please refer to Ketley et al. (2022).1


Highlights
Induce sequencespecific doublestrand breaks (DSBs) using U2OS-AsiSI-ER cells Target de novo transcribed RNA produced at DSBs with specifically designed FISH-PLA probes Detect the close proximity of a protein of interest to DSBderived RNA SUMMARY RNA transcribed at DNA double-strand breaks (DSBs) contributes to accurate DNA repair. Here, using the repair factors 53BP1 and TIRR as examples, we combine the fluorescence in situ hybridization (FISH) and proximity ligation assay (PLA) techniques to determine protein proximity to DSB-transcribed RNA. In this FISH-PLA protocol, we detail steps for designing DNA probes and image analysis using CellProfilerä software. This approach has many potential applications for the study of the RNA-binding proteins and nascent RNA interactions. For complete details on the use and execution of this protocol, please refer to Ketley 4. Culture cells, maintaining them in a transcriptionally active state (<70%-80% confluency).
Here, we described a modified PLA protocol using DNA oligonucleotide probes and a primary antibody against a protein of interest (POI) to detect proximity of de novo transcribed RNA and the POI upon DSB induction in the U2OS-AsiSI-ER cell line. The AsiSI restriction enzyme, linked to a modified ligand-binding domain of the estrogen receptor (ER), which translocates to the nucleus after the addition of 4-hydroxytamoxifen (4-OHT), allows the site/sequence-specific induction of the DSB, as measured by gH2AX levels 1,2 (Figures 1A and 1B).

Preparation of reagents
Timing: approximately 1 day 5. DEPC-H 2 O: Add 1 mL of DEPC to 1,000 mL of ddH 2 O in a screw-cap glass bottle. Incubate for 12-18 h at 20 C in a fume hood with swirling. Autoclave to inactivate DEPC. Store at 20 C. 6. RNAse-free buffers: prepare buffers using the autoclaved DEPC-H 2 O. Buffer recipes are shown in the materials and equipment section.
8. Optimization of the primary antibody: Antibody choice is critical to the success of the FISH-PLA assay. Generally, antibodies applicable for immunofluorescence experiments should work in the FISH-PLA setting. The working concentration of the primary antibody should be set starting from manufacturer's recommended concentration and titrated at different dilutions to establish specific signals vs background. The optimal primary antibody concentration could also be determined in a canonical dual antibody PLA assay.

MATERIALS AND EQUIPMENT
Basic laboratory materials such as a laminar culture hood, CO 2 incubator, microcentrifuge, glassware, and plasticware have not been mentioned in the tables below but are required for the protocol.

STEP-BY-STEP METHOD DETAILS
For an overview of the FISH-PLA workflow, see Figure 3.

Preparation of cells
Timing: 24 h The following steps describe the cell seeding procedure on poly-l-lysine coated coverslips in a 6 well plate format.
Note: The use of coating reagents such as poly-l-lysine is optional. Other coating reagents could also potentially be used such as Matrigelâ, gelatin, or collagen. CRITICAL: After poly-l-lysine incubation, wells should be washed at least twice with PBS as residual poly-l-lysine is toxic and may cause cell death. To ensure a successful FISH-PLA assay, cells should be maintained in a transcriptionally active state, and therefore the confluency should reach no more than 60%-70% after 24 h.

DSB induction, cell fixation, blocking
Timing: 6 h The steps below describe DSB induction, cell fixation and blocking.
CRITICAL: Before starting the DSB induction, switch on the hybridization oven and a thermoblock, setting the temperature at 95 C. Additionally, pre-warm a humidified chamber to 37 C.
6. Vacuum aspirate the culture media. 7. Add 500nM of 4-hydroxytamoxifen (4-OHT) in 2 mL of complete DMEM (13 Pen/Strep, 10% FBS) and incubate 37 C with 5% CO 2 for 4 h. 8. Wash twice with 2 mL ice cold PBS. 9. Add 2 mL of PFA (4% in PBS) and incubate for 15 min at room temperature to fix the cells, then wash 3 times with 2 mL ice cold PBS and permeabilize with 0.2%-0.5% Triton X100 for 10 min. 10. Wash 3 times with PBS. Add 80 mL of FISH-PLA Blocking Buffer (per coverslip) to a pre-warmed humidified chamber and place the coverslip face down on the FISH-PLA blocking solution. Incubate for 1 h at 37 C.

FISH-PLA DNA probes buffer preparation and oligonucleotide hybridization
Timing: 16-18 h The following steps summarize FISH-PLA probes hybridization, washing steps and primary antibody incubation.
11. FISH-PLA DNA probe buffer should be prepared in two steps. a. Firstly, add SSC, diluted DNA probes, and DEPC-H 2 O to an RNAse-free 1.5 mL tube.
Incubate at 95 C for 3 min followed by immediate snap-cooling on ice. b. After cooling, add Triton X-100, RNAsin plus, and sssDNA, keeping the solution on ice.
CRITICAL: The denaturation and snap-cooling steps are important to preserve the linearization of the DNA probes and avoid the formation of DNA secondary structure.
12. Add 60 mL of FISH-PLA probe buffer onto glass slides and place them in the humidified chamber.
Incubate in a hybridization oven at 95 C for 3 min, followed by 12-18 h incubation at 37 C.

FISH-PLA DNA probe washing
Timing: 30 min 13. Take the coverslip from the humidified chamber and place into new 6 well plate. Add 2 mL of 23 SSC washing buffer and incubate at 20 C for 5 min with gentle rocking. Repeat the SCC washing step twice more. 14. Wash twice with 2 mL PBS for 5 min, gently rocking. CRITICAL: Do not allow sample to dry out. To remove the excess buffer, quickly tap the coverslip on a tissue.

PLA Duolink protocol
Timing: 4 h The steps herein describe PLA part of the protocol using Duolink KIT components.

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Note: Prewarm PLA wash buffers A and B to room temperature in a water bath set at 37 C.  Pause point: The slides can be stored at 4 C in the dark. However, to ensure good image quality, it is recommended to image the slides within 3-4 days after mounting.

Confocal microscope setup
Timing: variable, mainly depends on the slide number and microscope-specific acquisition parameters (2-6 h) The steps below describe image acquisition and analysis using an Olympus Confocal Microscope and CellProfiler software.
CRITICAL: Sample slides should be imaged maintaining constant microscope parameters (e.g., laser power).
All imaging was performed using an Olympus Confocal Microscope FV1200, with a 603 objective. The filters DAPI (405nm) and Texas Red (559nm) were used, with speed settings at fast and an area of 1024 3 1024. The images were acquired using Z stacks at a 1 mm step size.

FISH-PLA image analysis using Fiji Image J and CellProfiler software
Timing: variable from 2-6 h CRITICAL: Confocal images acquired by the confocal microscope can be saved in oib format (Olympus Image Binary) and should be processed using Fiji Image J software. 38. Cellprofiler settings can be modified by selecting specific input modules at the top left of the CellProfiler window (i.e., Images and Name And Types), as described below. 39. Select the Images module and upload the images to be analyzed (e.g sample_name.oib -C = 0 and sample_name.oib -C = 1) by dragging and dropping the files into the Images tab. 40. Select the NameAndTypes module and select ''Images matching rules'' next to the ''assign a name to'' section.  For examples of the CellProfiler outputs see Figure 6.

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Note: To visualise the results of each module, select ''Start Test Mode'' located in the menu bar at bottom of the CellProfiler window. Pause and play icons will appear next to the different analysis modules. Select '' Step'' to visualize the outputs from each of the analysis modules ( Figure 4).
48. Finally in the section ''Export to Spreadsheet'', select the output folder and plot the Children Foci Count values present in the excel file ''Nuclei'' using GraphPad Prism software.
CRITICAL: For the accurate identification of both the nuclei and foci, some manual optimization in the input analysis settings may be required. Additional settings such as the threshold strategy, thresholding method, method to distinguish clumped objects, and method to draw dividing lines between clumped objects, can be optimised. In order to evaluate the results of the chosen settings, Test Mode can be used.

EXPECTED OUTCOMES
Roles of RNA-binding proteins in the DNA Damage Response (DDR) have been described. Moreover, different RNA species ranging from short non-coding RNAs (i.e., microRNAs (miRNAs), long non-coding RNAs (lncRNAs), DNA damage-dependent small RNAs (DDRNAs)), to long damage-induced and transcription-related RNAs (i.e., damage-responsive transcripts (DARTs), and damage-induced lncRNA (dilincRNAs)) have also been described as crucial players in RNAdependent DDR. 3 Traditional crosslinking-based and RNA-tagged approaches (e.g., crosslinked immunoprecipitation (CLIP) and RNA-protein interaction detection (RaPID)) have been successfully employed to identify and describe novel RNA-protein interactions. However, these techniques have several limitations including significant background noise due to a high level of non-specific RNAs, subcellular mislocalization of tagged RNAs, and sensitivity issues for low expressed RNAs. 4 Our FISH-PLA approach is a sensitive proximity ligation-based method for the proximal detection of sequence-specific RNA molecules and a protein of interest, overcoming some limitations associated with well-established assays for the detection RNA-protein interactions. The DNA probes used in this approach are a direct substrate for the PLA ligation step; this allows the detection of low abundant and transient RNA molecules, such as those produced upon DNA damage at DSBs.
We designed the DNA probes with strong and specific complementarity with the RNA of interest (50 nucleotides), with the addition of a linker of 41 deoxyadenosines that allows not only an efficient T4mediated ligation during the PLA ligation step, but also ensures the detection of RNA-protein proximity within the canonical 40 nm PLA proximity range. The probes also contain a 22 nucleotide sequence adapter important for the ligation step.
Furthermore, to reduce the chances of false-positive results, we included some experimental controls such as: the addition of an a-amanitin incubation step that reduces the presence of the de novo transcribed RNA molecules, the detection of the RNA-protein interaction at two different DSB loci (DS1 and DS2), the employment of a primary antibody against a well-known repair protein 53BP1, which has previously been shown to interact with de novo transcribed RNA, 5 and the use of siRNA knockdown and probe-only controls (Figure 7). It is also possible to use different experimental controls, such as scrambled DNA oligonucleotide probes.
This protocol could be used in a wide range of applications and can be extended to the detection and the validation of other RNA-protein interactions.

QUANTIFICATION AND STATISTICAL ANALYSIS
RNA-protein interactions were visualized using Fiji Image J software 6 and analyzed using CellProfilerä software 7 with the Speckle Counter pipeline. Statistical analysis was performed using GraphPad Prism 9 and the Mann Whitney test was performed.

LIMITATIONS
In the canonical PLA assay, DNA probes covalently linked to the secondary antibodies with specific sequences are important for the ligation step. The PLA minus probe consists of a secondary antibody linked to a DNA moiety of 10 adenosines, and a specific DNA sequence (5 0 GAC GCT AAT AGT TAA GAC GCT T) with three 3 0 -end 2 0 -O-methyl-RNA residues, while the PLA plus probe bears a DNA probe sequence characterized by a 10 adenosine linker and a DNA sequence 5 0 TAT GAC AGA ACT AGA CAC TCT T. The 2 0 -O-methyl-RNA residues are necessary to decrease the T4 DNA ligase-mediated ligation efficiency (by about 50%) to avoid spurious ligation resulting in false positive artefacts.
In this study we decided to use DNA probes bearing the specific sequence of the PLA plus probes, due to synthesis issues with long 108-mer DNA/RNA molecules.

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Nevertheless, this approach overcomes several issues of oligonucleotide-antibody PLA-based methods. For example, DNA probes are more stable compared to RNA probes and are not degraded by ubiquitous RNAses. Moreover, due to the higher thermodynamic stability of RNA:DNA hybrids over DNA:DNA duplexes, it is possible to ensure specific RNA hybridization through the use of sequential washes with incremental reduced salt concentration that enhance the hybridization stringency and RNA target recognition.
This may be related to high DNA probe concentrations and/or inappropriate antibody concentration.

Potential solution
Lower the DNA probe/antibody concentration and run experiment controls using DNA probes only and antibody only to check for non-specific signals.
Titrate the DNA probe concentration by performing FISH-PLA experiments using a-amanitin or Triptolide as controls.
Titrate the optimal antibody concentration by performing IF experiments with knockdowns as a control.
More stringent wash conditions should be considered by increasing the washing time and/or by adding more washing steps.

Problem 2
Low signal-to-noise ratio.
The source of this issue may depend on several steps: Fixation and permeabilization time. Drying out of the coverslips during the Duolink PLA washing.

Potential solution
Modify the PFA fixation time.
Keep the coverslip moist during the Duolink PLA washing. Add a pre-extraction step before fixation.
The absence of PLA signal may be caused by numerous reasons including: No 4-OHT-dependent DSB induction. Degradation of the target RNA. A low concentration of or poor quality DNA probes. Overconfluent cells at the time of the FISH-PLA experiment.

Potential solution
Check for efficient 4-OHT-mediated DSB induction by performing an IF experiment using the gH2A.X antibody.

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Use RNAse-free reagents and consumables. Make fresh DNA probes avoiding freeze-thaw cycles. Seed cells at a lower density.

Problem 4
Poor correlation between the imaged PLA foci and PLA foci numbers by CellProfiler automated counting.

Potential solution
To avoid inaccurate signal measurement, advance settings in the CellProfiler analysis modules can be adjusted. Run the Test Mode option to manually analyze the primary objects segmentation (Nuclei and Foci) and to verify the assigned count numbers of each object.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Monika Gullerova, monika.gullerova@path.ox.ac.uk.

Materials availability
Most materials required are commercially available.

Data and code availability
This protocol used CellProfiler software and code Speckle counter pipeline, both are available at www.cellprofiler.org.